The increasing prevalence of multidrug resistant infections caused by Gram-positive pathogens, such as methicillin resistant Staphylococcus aureus (MRSA), underscores the clinical need for novel strategies to treat life-threatening infections. New antibacterial drugs that are effective against MRSA have been introduced recently, but must be given intravenously, are limited to hospital use, are not tolerated well, and are already subject to resistance. The overall goal of the proposed research is discover a novel class of drugs that target the wall teichoic acid (WTA) biosynthetic pathway of S. aureus. WTAs are phosphate rich, highly anionic polymers that are covalently linked to the cell wall of most Gram-positive pathogens. Inactivation of the later enzymatic steps of the WTA pathway prevents growth, unless the pathway is blocked by inactivation of the initial enzymatic steps. Loss of WTA in S. aureus results in abnormalities in cell wall structure and cell division, and increases autolysis. WTA also plays an important role in the following aspects of virulence: host colonization, resistance to antibacterial peptides and lipids, and biofilm formation. Significantly, loss of WTA increases the sensitivity of MRSA to beta-lactam antibiotics. Potent WTA inhibitors will provide multiple therapeutic benefits, such as bacterial growth inhibition, decreased virulence, and renewed clinical utility of beta- lactam antibiotics for treating MRSA, which represents a highly significant innovation. Our strategy is to identify potent inhibitors of specific steps in the WTA biosynthetic pathway, and to develop them into antibacterial drugs that will be used in combination therapy with an FDA-approved beta-lactam antibiotic to treat S. aureus infections. In Phase I, we will modify and optimize a validated screening assay to increase the sensitivity of the assay for inhibitors of specific WTA targets. We will use this assay to screen >300,000 discreet small molecule compounds to identify inhibitors, and we will prioritize them using a panel of existing secondary assays designed to evaluate antibacterial potency, antibacterial spectrum, selectivity, frequency of resistance, and potentiation of beta-lactam antibiotics. We will verify the molecular target of high priority hits. In Phase II, we will develop the most promising validated hits into lead compounds by optimizing their activity and specificity using rational drug design. The specific aims of this proposal are as follows. Aim 1. Modify and optimize an established cell based, pathway specific screening assay to favor the identification of inhibitors of specific steps of WTA biosynthesis (TarB, D, and F). Aim 2. Screen a diverse compound library, identify, and confirm inhibitors of WTA synthesis. Aim 3. Validate confirmed inhibitors of WTA synthesis using secondary assays and identify hit series. Aim 4. Verify mechanism of action of selected hit series.